WO2019184025A1

WO2019184025A1 - Method for preparing polyarabogalacturonic acid by using dried tangerine peel

Info

Publication number: WO2019184025A1
Application number: PCT/CN2018/083829
Authority: WO
Inventors: 杨宝
Original assignee: 中国科学院华南植物园
Priority date: 2018-03-26
Filing date: 2018-04-20
Publication date: 2019-10-03
Also published as: US20200317822A1; CN108546304A

Abstract

Disclosed is a method for preparing polyarabogalacturonic acid by using dried tangerine peel. The dried tangerine peel is subjected to pulverization, leaching by adding water, and separation, and a leaching liquid is collected, and then concentrated, ethanol is added to precipitate a polysaccharide substance, the precipitate is collected to obtain a crude polysaccharide of dried tangerine peel, the crude polysaccharide of dried tangerine peel is purified by means of an anion exchange column, and gradient eluted using water and an NaCl solution, a component eluted from the NaCl solution is collected, same is concentrated and then continues to be purified by a gel filtration column, and eluted using water, and the target component is collected, and concentrated to dryness to obtain the polyarabogalacturonic acid. The method prepares the polyarabogalacturonic acid from dried tangerine peel and separates out same, with a yield of 6-45 g/kg (a purity of 80%-95%).

Description

Method for preparing polyarabinogalacturonan by using tangerine peel

Technical field:

The invention belongs to the field of agricultural product processing, and particularly relates to a method for preparing polyarabinogalacturonan by using tangerine peel.

Background technique:

The dried tangerine peel is the mature fruit of the Citrus reticulata Blanco, which is mainly produced in many provinces in southern China. Chenpi is an important traditional Chinese herbal medicine, and it is also a food and food homologous food. It can treat vomiting, improve gastrointestinal function and promote digestion. It has a good effect on cardiovascular diseases and can effectively lower blood lipids and blood pressure. In addition, there are antibacterial, anti-inflammatory, anti-inflammatory and other effects. Used in cooking seasoning, tangerine peel can remove the astringency and enhance the aroma of the food. At present, reports on the active substances of tangerine peel are concentrated on aromatic essential oils, methoxy flavonoids, etc., and the structural characteristics of the main polysaccharide components have not yet been identified. Therefore, it is necessary to carry out research on the separation, purification and structure identification of dried tangerine peel polysaccharides, revealing the structure of the main polysaccharides, which is of great significance for the definition of the chemical composition of dried tangerine peel.

Summary of the invention:

It is an object of the present invention to provide a process for the preparation of polyarabinogalacturonan using dried tangerine peel.

The invention finds and prepares a main polysaccharide in the tangerine peel, and the structure is determined to be polyarabinogalacturonic acid by NMR analysis, and the chemical structure thereof is as shown in the formula (I):

Wherein R is hydrogen or a methyl ester group.

In general, GalpA in formula (I) is an abbreviation for galacturonic acid, namely galacturonic acid; Araf refers to arabinose, ie arabinose.

The method for preparing polyarabinogalacturonic acid of the present invention, comprising the steps of:

The dried tangerine peel is pulverized, extracted with water, separated and collected, and then concentrated. The polysaccharide is precipitated by adding ethanol to collect the precipitate, and the crude polysaccharide of dried tangerine peel is obtained. The crude polysaccharide of dried tangerine peel is purified by anion exchange column, and washed with water and NaCl solution. The component was eluted by collecting the NaCl solution, concentrated, and then purified by a gel filtration column, eluted with water, and the target component was collected, and concentrated to obtain polyarabinogalacturonic acid.

Preferably, the method comprises the steps of: purifying the crude polysaccharide of the tangerine peel by using an anion exchange column, eluting with a gradient of water and NaCl solution, collecting the eluted component of the NaCl solution, concentrating, purifying by a gel filtration column, eluting with water, collecting the target component, The concentrated arabic galacturonic acid is obtained by purifying the crude polysaccharide of dried tangerine peel by anion exchange column, first eluting with water to remove the neutral polysaccharide component, and then eluting with NaCl solution, collecting the eluted component, concentrating and condensing The gel filtration column was purified, and eluted with water as a solvent, and the target polysaccharide component was collected, concentrated, and dried to obtain polyarabinogalacturonic acid.

Preferably, the dried tangerine peel is pulverized and water leaching is carried out by pulverizing the dried tangerine peel, adding 1 to 30 times of mass of water, and leaching at 25 to 100 ° C for 1 to 120 hours.

Preferably, the separating and collecting the separation in the extract is by centrifugation or filtration.

Preferably, the ethanol is added for precipitation by adding ethanol to the final concentration of the extract to a volume fraction of 20% to 90% for precipitation.

The dried tangerine peel is fresh tangerine peel or dried tangerine peel.

The NaCl solution is preferably a 0.2 M NaCl aqueous solution.

The invention also provides the use of dried tangerine peel in the preparation of polyaragal galacturonic acid.

The polyarabinogalacturonan obtained by the present invention has good immunopotentiating activity.

The present invention is prepared by separating and obtaining polyarabinogalacturonic acid from dried tangerine peel, and the yield thereof is 6 to 45 g/kg (purity is 80 to 95%). The invention provides a new method for the preparation of polyarabinogalacturonan, which is of great significance for promoting the deep processing and utilization of dried tangerine peel, enhancing the added value of tangerine peel products and promoting the sustainable development of the industry.

detailed description

The following examples are intended to further illustrate the invention and not to limit the invention.

Example 1: Preparation and Structure Identification of Polyarabinogalacturonic Acid

1. Preparation and separation of polyarabinogalacturonic acid

1) Material selection: choose fresh dried tangerine peel and crush;

2) Leaching: adding 15 times of water of dried tangerine peel, leaching at 50 ° C for 2 hours, centrifuging, collecting the extract;

3) Precipitation of organic solvent: The above extract was concentrated, and ethanol was added to a final concentration of 60% by volume (extraction solution: ethanol = 60:40, v/v), and left at 4 ° C for 12 hours. Centrifugation, collecting the precipitate, which is the crude polysaccharide of dried tangerine peel;

4) Column purification: crude tangerine peel polysaccharide was purified by DEAE Sepharose Fast Flow anion exchange column, eluted with water/NaCl solution, eluted with water, and then eluted with 0.2M NaCl solution to collect the components eluted by NaCl solution. After concentration, the Sephadex G100 gel filtration column (15×460 mm) was eluted with water at a flow rate of 0.5 ml/min, and the polysaccharide component which was eluted for 50 to 160 minutes was collected, and dried to obtain polysaccharide A (polygalactose). Aldehyde acid).

The yield of polyarabinogalacturonan obtained by this method is 26 to 35 g/kg, and the purity is 80 to 95%.

2. Structure identification of polysaccharide A (polyarabinogalacturonic acid)

Polysaccharide A (polyarabinogalacturonan) was soluble in water, and the results of ¹ H NMR (500 MHz, D ₂ O) and ¹³ C NMR (125 MHz) of the polysaccharide are shown in Table 1.

Table 1. Chemical shifts of ¹³ C and ¹ H spectra of tangerine peel arabinogalacturonic acid

According to the NMR information, the structure of the polysaccharide was identified as shown in the formula (I), and the name was polyarabinogalacturonic acid.

Wherein R is hydrogen or a methyl ester group.

Example 2:

1) Material selection: choose fresh dried tangerine peel and crush;

2) leaching: adding 1 times of water of dried tangerine peel, leaching for 120 hours at room temperature (25 ° C), filtering, collecting the extract;

3) Precipitation of organic solvent: The above extract was concentrated, and ethanol was added to a final concentration of 50% by volume (extraction solution: ethanol = 50:50, v/v), and left at 4 ° C for 12 hours. Centrifugation, collecting the precipitate, which is the crude polysaccharide of dried tangerine peel;

4) Column purification: crude tangerine peel polysaccharide was purified by DEAE Sepharose Fast Flow anion exchange column, eluted with water/NaCl solution, eluted with water, and then eluted with 0.2M NaCl solution to collect the components eluted by NaCl solution. After concentration, the Sephadex G100 gel filtration column was applied, and eluted with water to collect the target polysaccharide component, which was concentrated and dried to obtain polysaccharide A (polyarabinogalacturonan). The structure was identified as in Example 1.

The polyarabinogalacturonan obtained by this method has a yield of 6 to 16 g/kg and a purity of 80 to 95%.

Example 3:

1) Material selection: choose fresh dried tangerine peel and crush;

2) leaching: adding 30 times of water of dried tangerine peel, leaching at 100 ° C for 5 hours, filtering, collecting the extract;

3) Precipitation of organic solvent: The above extract was concentrated, and ethanol was added to a final concentration of 90% by volume (extraction solution: ethanol = 90:10, v/v), and left at 4 ° C for 12 hours. Centrifugation, collecting the precipitate, which is the crude polysaccharide of dried tangerine peel;

The yield of polyarabinogalacturonan obtained by this method is 32 to 45 g/kg, and the purity is 80 to 95%.

Example 4:

1) Material selection: choose dried tangerine peel and smash;

2) leaching: adding 30 times of water of dried tangerine peel, leaching at 60 ° C for 1 hour, filtering, collecting the extract;

3) Precipitation of organic solvent: The above extract was concentrated, and ethanol was added to a final concentration of 20% by volume (extraction solution: ethanol = 20:80, v/v), and left at 4 ° C for 12 hours. Centrifugation, collecting the precipitate, which is the crude polysaccharide of dried tangerine peel;

The polyarabinogalacturonan obtained by this method has a yield of 11 to 23 g/kg and a purity of 80 to 95%.

Claims

Polyaragalacturonan having a chemical structure as shown in formula (I):

Wherein R is hydrogen or a methyl ester group.
A method of producing the polyaragal galacturonic acid according to claim 1, wherein the polyaragal galacturonic acid is isolated from dried tangerine peel.
The method according to claim 2, wherein the dried tangerine peel is pulverized, water is extracted, the extract is separated and collected, and then concentrated, and ethanol is added to precipitate a polysaccharide, and the precipitate is collected to obtain a crude polysaccharide of the tangerine peel, using an anion. Purification of crude tangerine peel by exchange column, elution with water and NaCl solution, collecting the eluted fraction of NaCl solution, concentrating and purifying by gel filtration column, eluting with water, collecting the target components, and concentrating and drying to obtain polyarabinogalactose. acid.
The method according to claim 3, wherein said "purifying the crude orange peel with an anion exchange column, eluting with a gradient of water and NaCl solution, collecting the eluted components of the NaCl solution, and concentrating and continuing to use the gel filtration column. Purification, water elution, collection of target components, concentration and drying to obtain polyarabinogalacturonic acid" is to purify the crude polysaccharide of dried tangerine peel by anion exchange column, first elute with water to remove the neutral polysaccharide component, and then elute with NaCl solution. The eluted fraction was collected, concentrated, purified by a gel filtration column, eluted with water as a solvent, and the target polysaccharide component was collected, concentrated, and dried to obtain polyarabinogalacturonic acid.
The method according to claim 3, wherein the rinsing of the tangerine peel and the water leaching are carried out by pulverizing the tangerine peel, adding 1 to 30 times of mass of water, and leaching at 25 to 100 ° C for 1 to 120 hours.
The method of claim 3 wherein said separating and collecting the separation in the leachate is by centrifugation or filtration.
The method according to claim 3, wherein said adding ethanol precipitate is performed by adding ethanol to the final concentration of the extract in a volume fraction of 20% to 90%.
The method according to claim 3, wherein the dried tangerine peel is fresh tangerine peel or dried tangerine peel.
The method of claim 3 wherein said NaCl solution is a 0.2 M aqueous solution of NaCl.
The application of dried tangerine peel in the preparation of polyaragal galacturonic acid.